A two-pronged defense against bacterial self-intoxication

Researchers solved the structure of a bacterial toxin bound to a neutralizing protein, revealing two distinct mechanisms for how the toxin-producing bacteria avoid poisoning themselves.

Microbial communities are of fundamental importance to virtually all natural ecosystems, from the ocean floor to the gastrointestinal tract. Although the term “communities” implies cooperation, scientists now realize that bacterial colonies compete with each other for life-sustaining resources, availing themselves of a variety of strategies to reduce overcrowding. In some cases, they secrete toxins in their fight for survival. Here, researchers studied one such toxin from the bacterium Serratia proteamaculans, various strains of which live inside tree roots or inhabit the digestive tracts of insects and other animals.

Toxin targets cell division

The researchers showed that the toxin, Tre1, targets a bacterial protein, FtsZ, which is analogous to tubulin in human cells. Tubulin molecules are the building blocks of microtubules—long polymers that provide structure and shape to our cells and play an important role in cell division. In bacteria, FtsZ loses the ability to polymerize when attacked by the Tre1 toxin. Instead of dividing, the intoxicated cells grow longer and longer until they eventually split open and die (cellular elongation and lysis).

>Read more on the Advanced Light Source website

Image: Healthy bacteria (left) and bacteria (right) whose cell-division machinery has been disrupted by a toxin newly discovered in some bacterial arsenals.
Credit: Mougous Lab

Understanding the protein responsible for regulating heartbeats

A new research project uses the Canadian Light Source to help researchers understand the protein responsible for regulating heartbeats. Errors in this crucial protein’s structure can lead to potentially deadly arrhythmias, and understanding its structure should help researchers develop treatments. This protein, calmodulin (CaM), regulates the signals that cause the heart to contract and relax in almost all animals with a heartbeat.

“Usually you find some differences between versions of proteins from one species to another,” explains Filip Van Petegem, a professor in the University of British Columbia’s Department of Biochemistry and Molecular Biology. “For calmodulin that’s not the case—it’s so incredibly conserved.”

It also oversees hundreds of different proteins within the body, adjusting a broad array of cellular functions that are as crucial to our survival and health as a steady heartbeat.

>Read more on the Canadian Light Source website

Image: A surface representation of the disease mutant CaM (D95V, red) in complex with the piece of the voltage-gated calcium channel (blue).

Know your ennemy

Light source identifies a key protein interaction during E. coli infection

Escherichia coli is a common source for contaminated water and food products, causing the condition known as gastroenteritis with symptoms that include diarrhea, vomiting, fever, loss of energy, and dehydration. In fact, for children or individuals with weakened immune systems, this bacterial infection in the gut can be life-threatening.

One of the microbes responsible for gastroenteritis, known formally as enteropathogenic E. coli (EPEC), causes infections by directing a pointed, needle-like projection into the human intestinal tract, releasing toxins that make people sick.

“Enteropathogenic E. coli can fire toxic proteins from inside the bacterium right into the cells of your gut lining,” says Dustin Little, a post-doctoral researcher in the Brian Coombes lab at McMaster University’s Department of Biochemistry and Biomedical Sciences.

>Read more on the Canadian Light Source website

Image: Dustin Little and Brian Coombes in the lab.
Credit: Dustin Little. 

SwissFEL makes protein structures visible

Successful pilot experiment on biomolecules at the newest large research facility of PSI

For the development of new medicinal agents, accurate knowledge of biological processes in the body is a prerequisite. Here proteins play a crucial role. At the Paul Scherrer Institute PSI, the X-ray free-electron laser SwissFEL has now, for the first time, directed its strong light onto protein crystals and made their structures visible. The special characteristics of the X-ray laser enable completely novel experiments in which scientists can watch how proteins move and change their shape. The new method, which in Switzerland is only possible at PSI, will in the future aid in the discovery of new drugs.

Less than two years after the X-ray free-electron laser SwissFEL started operations, PSI researchers, together with the Swiss company leadXpro, have successfully completed their first experiment using it to study biological molecules. With that, they have achieved another milestone before this new PSI large research facility becomes available for experiments, at the beginning of 2019, to all users from academia and industry. SwissFEL is one of only five facilities worldwide in which researchers can investigate biological processes in proteins or protein complexes with high-energy X-ray laser light.

>Read more on the SwissFEL website

Image: Michael Hennig (left) and Karol Nass at the experiment station in SwissFEL where their pilot experiment was conducted.
Credit: Paul Scherrer Institute/Mahir Dzambegovic

Year of Engineering I23 Gripper Spotlight

Celebrating the Year of Engineering on Beamline I23

The Year of Engineering (UK) is all about celebrating the world and wonder of the industry, and exploring the wide range of ideas and innovations that Engineering involves. Today, we’re having a look at Diamond’s Beamline I23 – a specially designed instrument for protein crystallography that uses long wavelengths.
There are unique engineering scientific challenges involved in designing a system that will allow researchers to use long wavelengths of Synchrotron radiation effectively. The special cryogenically-cooled sample gripper on I23, is one of the solutions that makes this beamline successful. Learn more about this engineering innovation.

>Read more and watch more videos on the Diamond Light Source website

First European XFEL research results published

High number of X-ray pulses per second reduces time needed for the study of biological structures.

Just days before the first anniversary of the start of European XFEL user operation, the first results based on research performed at the facility have been published. In the journal Nature Communications, the scientists, headed by Prof. Ilme Schlichting from Max-Planck-Institute for Medical Research in Heidelberg, Germany, together with colleagues from Rutgers State University of New Jersey, USA, France, DESY and European XFEL, describe their work using the intense X-ray laser beam to determine the 3D structure of several proteins. They demonstrate, for the first time that, under the conditions used at the time of the experiment an increased number of X-ray pulses per second as produced by the European XFEL can be successfully used to determine the structure of biomolecules. As much faster data collection is therefore possible, the time needed for an experiment could be significantly shortened. The detailed determination of the 3D structure of biomolecules is crucial for providing insights into informing the development of  novel drugs to treat diseases.

Prof. Ilme Schlichting said: “Our work shows that under the conditions used data can be collected at European XFEL at a rate much faster than has ever been previously possible. As the time and cost of experiments decrease, very soon many more researchers will be able to perform experiments at high repetition rate X-ray lasers. Our results are therefore of interest not only tor the fields of biology and medicine, but also physics, chemistry and other disciplines.”

>Read more on the European XFEL website

Image: Guest scientist Tokushi Sato working at the sample chamber of the SPB/SFX instrument.
Credit: European XFEL

Structure reveals mechanism behind periodic paralysis

The results suggest possible drug designs that could provide relief to patients with a genetic disorder that causes them to be overcome suddenly with profound muscle weakness.

A rare genetic disorder called hypokalemic periodic paralysis (hypoPP) causes sudden, profound muscle weakness in people who occasionally exhibit low levels of potassium in their blood, or hypokalemia. When a patient is hypokalemic, hypoPP affects the function of the muscles responsible for skeletal movement. The disease has been known to stem from mutations in certain membrane proteins that channel and regulate the flow of sodium into cells. Exactly how the mutation affects the proteins’ function, however, was not known.

In earlier work, researchers from the Catterall Lab at the University of Washington had solved the structure of a sodium channel called NavAb from a prokaryote (single-celled organism). As a next step, the group decided to see if NavAb could serve as a model for studying the mutations that cause hypoPP in humans (eukaryotes), with the goal of finding a way to prevent or treat this disorder.

A leak in the pipe?

In a resting state, muscle-cell membranes keep potassium ions and sodium ions separated, inside and outside the cell, respectively, creating a voltage across the membrane. A chemical signal from a nerve cell sets off a cascade of events that results in sodium ions flowing into the cell, changing the membrane potential and and ultimately triggering muscle contraction.

>Read more on the Advanced Light Source website

Image: Three states of the voltage-sensing domain (VSD) of a membrane-channel protein. In the normal state, the water-accessible space (magenta) does not extend through the channel, preventing sodium (gray spheres) from passing through. In the disease state, a clear passage allows sodium to leak through, resulting in muscle paralysis. In the “rescued” state, the binding of guanidinium (blue and yellow spheres) effectively closes the channel and blocks sodium leakage. The red sphere represents the location of the disease-causing mutation. The side-chain sticks represent the voltage sensors of the sodium channel.

Open and shut: pain signals in nerve cells

Our daily function depends on signals traveling between nerve cells (neurons) along fine-tuned pathways. Central nervous system neurons contain acid-sensing ion channel 1a (ASIC1a), a protein important in sensing pain and forming memories of fear. An ion channel lodged in the cell membrane that provides a pathway for sodium ions to enter the cell, ASIC1a opens and closes in response to changes in extracellular proton concentrations. When protons accumulate outside the neuron, the channel opens, allowing sodium ions to flow into the cell, depolarizing the cell membrane and generating an electrical signal. The channel eventually becomes desensitized to protons and the gate closes. Scientists have visualized both the open and desensitized channel structures, but the third structure, which forms when the protons dissipate and the channel closes, remained elusive. Using protein crystallography at the ALS, researchers finally visualized the closed channel.

>Read more on the Advanced Light Source website

Animation: As the proton concentration increases or decreases, the gated channel ASIC1a toggles between open and closed positions, controlling the timing of signals traveling through the cell membrane of one neuron en route to the next.

How legionella manipulates the host cell by means of molecular mimics

Using synchrotron light, researchers from CIC bioGUNE have solved the structure of RavN, a protein that Legionella pneumophila uses for stealing functions and resources of the host cell.

Mimicry is the ability of some animals to resemble others in their environment to ensure their survival. A classic example is the stick bug whose shape and colour make him unnoticed to possible predators. Many intracellular pathogens also use molecular mimicry to ensure their survival. A part of a protein of the pathogen resembles another protein totally different from the host and many intracellular microorganisms use this capability to interfere in cellular processes that enable their survival and replication.

The Membrane Trafficking laboratory of the CIC bioGUNE in the Basque Country, led by Aitor Hierro, in collaboration with other groups from the National Institutes of Health in the United States, have been working for several years in understanding how the infectious bacterium Legionella pneumhopila interacts with human cells. During this research, experiments have been carried out at the XALOC beamline of the ALBA Synchrotron and I04 beamline of Diamond Light Source (UK). The results enabled scientists to solve the structure of RavN, a protein of L. pneumophila that uses this molecular mimicry to trick the infected cell.

>Read more on the ALBA website

Figure: (extract) Schematic representation of the structure of RavN1-123 as ribbon diagram displayed in two orientations (rotated by 90° along the x axis). Secondary elements are indicated as spirals (helices) or arrows (beta strands), with the RING/U-box motif colored in orange and the C-terminal structure colored in slate. (Full image here)

First serial crystallography experiments performed at BioMAX

BioMAX has successfully performed the first serial crystallography experiments at the beamline. This new method is performed at room temperature which allows structural biologists to study their molecules at more biologically relevant conditions. The technique can also be used on smaller crystals which will alleviate some of the restrictions for molecules such as membrane proteins, that do not typically form large crystals. Eventually, it is hoped that this technique will allow users at the BioMAX and MicroMAX beamlines to take snapshots of the dynamic states of proteins in rapid succession giving a dynamic view of protein movement and activity.

The serial crystallography technique promises to be very useful to users of both synchrotrons and XFELs. Over the course of one experiment, users were able to measure between 20 and 50 crystals every second, resulting in 20 TB of data from just 3 proteins. BioMAX hopes to quickly master this complex technique in order to offer it to users as soon as possible. It also gives us a glimpse of what will be possible at the newly funded MicroMAX beamline.

>Read more on the MAX IV Laboratory website

Image: BioMAX serial crystallography setup using a High Viscosity Extrusion (HVE) injector specially designed for the BioMAX endstation by Bruce Doak of the Max Planck Institute for Medical Research, Heidelberg, and fabricated at that institute.

ALS passes the 7000-protein milestone

The eight structural biology beamlines at the ALS have now collectively deposited over 7000 proteins into the Protein Data Bank (PDB), a worldwide, open-access repository of protein structures. The 7000th ALS protein structure (entry no. 6C7C) is an enzyme from Mycobacterium ulcerans (strain Agy99), solved with data from Beamline 5.0.2. This bacterium produces a toxin that eats away at skin tissue, causing what’s known as Buruli ulcers (Google at your own risk!). The bacterium is antibiotic-resistant, and treatment involves the surgical removal of infected tissues, including amputation.

The enzyme structure was solved by a group from the Seattle Structural Genomics Center for Infectious Disease (SSGID), whose mission is to obtain crystal structures of potential drug targets on the priority pathogen list of the National Institute of Allergy and Infectious Diseases (NIAID). As of May 2018, SSGCID has deposited 1090 structures in the PDB, with data for more than a quarter of those collected at ALS beamlines.

>Read more on the Advanced Light Source website

Image: PDB 6C7C: Enoyl-CoA hydratase, an enzyme from M. ulcerans (strain Agy99).

New high-precision instrument enables rapid measurements of protein crystals

A team of scientists and engineers at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have developed a new scientific instrument that enables ultra-precise and high-speed characterization of protein crystals at the National Synchrotron Light Source II (NSLS-II)—a DOE Office of Science User Facility at Brookhaven, which generates high energy x-rays that can be harnessed to probe the protein crystals. Called the FastForward MX goniometer, this advanced instrument will significantly increase the efficiency of protein crystallography by reducing the run time of experiments from hours to minutes.

Protein crystallography is an essential research technique that uses x-ray diffraction for uncovering the 3D structures of proteins and other complex biological molecules, and understanding their function within our cells. Using this knowledge about the basic structure of life, scientists can advance drug design, improve medical treatments, and unravel other environmental and biochemical processes governing our everyday lives.

>Read more on the NSLS-II website

Image: Yuan Gao, Wuxian Shi, Evgeny Nazaretski, Stuart Myers, Weihe Xu and, Martin Fuchs designed and implemented the new goniometer scanner system for ultra-fast and efficient serial protein crystallography at the Frontier Microfocusing Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II.

Respiratory virus study points to likely vaccine target

X-ray laser opens new view on Alzheimer proteins

Graphene enables structural analysis of naturally occurring amyloids

A new experimental method permits the X-ray analysis of amyloids, a class of large, filamentous biomolecules which are an important hallmark of diseases such as Alzheimer’s and Parkinson’s. An international team of researchers headed by DESY scientists has used a powerful X-ray laser to gain insights into the structure of different amyloid samples. The X-ray scattering from amyloid fibrils give patterns somewhat similar to those obtained by Rosalind Franklin from DNA in 1952, which led to the discovery of the well-known structure, the double helix. The X-ray laser, trillions of times more intense than Franklin’s X-ray tube, opens up the ability to examine individual amyloid fibrils, the constituents of amyloid filaments. With such powerful X-ray beams any extraneous material can overwhelm the signal from the invisibly small fibril sample. Ultrathin carbon film – graphene – solved this problem to allow extremely sensitive patterns to be recorded. This marks an important step towards studying individual molecules using X-ray lasers, a goal that structural biologists have long been pursuing. The scientists present their new technique in the journal Nature Communications.

Amyloids are long, ordered strands of proteins which consist of thousands of identical subunits. While amyloids are believed to play a major role in the development of neurodegenerative diseases, recently more and more functional amyloid forms have been identified. “The ‘feel-good hormone’ endorphin, for example, can form amyloid fibrils in the pituitary gland. They dissolve into individual molecules when the acidity of their surroundings changes, after which these molecules can fulfil their purpose in the body,” explains DESY’s Carolin Seuring, a scientist at the Center for Free-Electron Laser Science (CFEL) and the principal author of the paper. “Other amyloid proteins, such as those found in post-mortem brains of patients suffering from Alzheimer’s, accumulate as amyloid fibrils in the brain, and cannot be broken down and therefore impair brain function in the long term.”

Image: On the ultra-thin, extremely regular layer of graphene, the fibrils align themselves in parallel in large domains. The intense X-ray light from the X-rax free-electron laser LCLS at the SLAC National Accelerator Center enabled the researchers to gain partial information about the fibril structure from ensembles of just a few fibrils.
Credit: Greg Stewart/SLAC National Accelerator Laboratory

The proteins that bind

Researchers reveal the structure of a protein that helps bacteria aggregate

Serine-rich repeat proteins (SRRPs), which help bacteria attach to surfaces, have been structurally characterised in pathogenic bacteria but not in beneficial bacteria such as those present in the gut. Dr Nathalie Juge’s team at the Quadram Institute Bioscience has previously identified SRRP as a main adhesin in Lactobacillus reuteri strains from pigs and mice. Now, together with colleagues at the University of East Anglia, they have described the structure and activity of the binding region of L. reuteri SRRPs in a paper published in PNAS. Using the Macromolecular Crystallography beamlines (I03 and I04) at Diamond Light Source, they discovered that the structure of these proteins is unique among characterised SRRPs and is surprisingly similar to pectin degrading enzymes. Molecular simulations and binding experiments revealed a pH-dependent binding to pectin and to proteins from the epithelium known as mucins. Altogether, these findings shed light on the activity of a key protein in these bacteria and may help guide the development of more targeted probiotic interventions.

>Read more on the Diamond Light Source website

Figure: (Left) Cartoon representation of crystal structures of the binding region of SRRP53608. (Right) Cartoon representation of crystal structures of the binding region of SRRP100-23. The N-terminus is shown with blue balls and the C-terminus is shown with red balls.

Discovery of the novel green fluorescent protein by NSRRC

Scientist Dr. Chun-Jung Chen and Research Assistant Mr. Yen-Chieh Huang of the National Synchrotron Radiation Research Center collaborated with the researchers at the University of the Philippines – Diliman to analyze the three-dimensional structure and functional characteristics of the novel green fluorescent protein asFP504 isolated from a soft coral species, Alcyonium sp. found at the Taklong Island, Guimaras, Philippines. The results of the study were published and selected as the cover story on the Philippine Journal of Science in March, which is considered as one of the representative research results of the Southward Policy of NSRRC.

>Read more on the National Synchrotron Radiation Research Center (NSSRC) website

Image: Extract of the cover on the Philippine Journal of Science (2018.03)